OBJECTIVE

Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another.

METHODS

Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles.

RESULTS

SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold).

CONCLUSIONS

The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.

Hepatocyte growth factor (HGF) is a humoral mediator of epithelial-mesenchymal interactions, acting on a variety of epithelial cells as mitogen, motogen, and morphogen. Exogenous HGF acts as a hepatotrophic factor and a renotrophic factor during experimental injury. To investigate whether HGF has a pulmotrophic function, human recombinant HGF was administered to C57BL/6 mice with severe lung injury by bleomycin (BLM). Low dose simultaneous and continuous administration of HGF (50 micrograms/mouse/7 d) with BLM (100 mg/mouse/7 d) repressed fibrotic morphological changes at 2 and 4 wk. Ashcroft score showed a significant difference in lung fibrosis with and without HGF at 4 wk (3.7 +/- 0.4 versus 4.9 +/- 0.3, p < 0.05). Furthermore, either simultaneous or delayed administration of high dose HGF (280 micrograms/mouse/14 d) equally repressed fibrotic changes by BLM when examined at 4 wk (Ashcroft score: 2.6 +/- 0.4 and 2.4 +/- 0.2 versus 4.1 +/- 0.2, p < 0.01). Hydroxyproline content in the lungs was significantly lower in mice with either simultaneous or delayed administration of high dose HGF as compared to those administered BLM alone (121.8 +/- 8.1% and 113.2 +/- 6.2% versus 162.7 +/- 4.6%, p < 0.001). These findings indicate that exogenous HGF acts as a pulmotrophic factor in vivo and prevents the progression of BLM-induced lung injury when administered in either a simultaneous or delayed fashion. HGF may be a potent candidate to prevent or treat lung fibrosis.

OBJECTIVE

Keratoconus is a progressive disorder of the cornea that can lead to severe visual impairment or blindness. Although several genomic regions have been linked to rare familial forms of keratoconus, no genes have yet been definitively identified for common forms of the disease.

METHODS

Two genome-wide association scans were undertaken in parallel. The first used pooled DNA from an Australian cohort, followed by typing of top-ranked single-nucleotide polymorphisms (SNPs) in individual DNA samples. The second was conducted in individually genotyped patients, and controls from the USA. Tag SNPs around the hepatocyte growth factor (HGF) gene were typed in three additional replication cohorts. Serum levels of HGF protein in normal individuals were assessed with ELISA and correlated with genotype.

RESULTS

The only SNP observed to be associated in both the pooled discovery and primary replication cohort was rs1014091, located upstream of the HGF gene. The nearby SNP rs3735520 was found to be associated in the individually typed discovery cohort (P = 6.1 × 10(-7)). Genotyping of tag SNPs around HGF revealed association at rs3735520 and rs17501108/rs1014091 in four of the five cohorts. Meta-analysis of all five datasets together yielded suggestive P values for rs3735520 (P = 9.9 × 10(-7)) and rs17501108 (P = 9.9 × 10(-5)). In addition, SNP rs3735520 was found to be associated with serum HGF level in normal individuals (P = 0.036).

CONCLUSIONS

Taken together, these results implicate genetic variation at the HGF locus with keratoconus susceptibility.

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor c-met have been implicated in the genesis, malignant progression, and chemo/radioresistance of multiple human malignancies, including gliomas. We examined the antitumor effects of targeting SF/HGF and c-met expression in pre-established glioma xenografts by using novel chimeric U1snRNA/ribozymes. Transient expression of anti-SF/HGF and anti-c-met U1snRNA/ribozymes inhibited SF/HGF and c-met expression, c-met receptor activation, tumor cell migration, and anchorage-independent colony formation in vitro. Delivery of U1snRNA/ribozymes to established subcutaneous glioma xenografts via liposome-DNA complexes significantly inhibited tumor growth as well as tumor SF/HGF and c-met expression levels. Histologic analysis of tumors treated with U1snRNA/ribozymes showed a significant decrease in blood vessel density, an increase in activation of the pro-apoptotic enzyme caspase-3, and an increase in tumor cell apoptosis. Treatment of animals bearing intracranial glioma xenografts with anti-SF/HGF and anti-c-met U1snRNA/ribozymes by either intratumoral injections of adenoviruses expressing the transgenes or intravenous injections of U1snRNA/ribozyme-liposome complexes substantially inhibited tumor growth and promoted animal survival. We demonstrate that SF/HGF and/or c-met expression can be targeted in vivo to inhibit tumor growth. In addition, our findings represent the first in vivo application of chimeric U1snRNA/ribozymes, which have numerous potential therapeutic gene-targeting applications.

Growth factors such as hepatocyte growth factor (HGF) are highly up-regulated during development and following renal injury and are known to induce marked morphogenic actions in cultured tubular epithelial cells, including scattering, migration, single cell branching morphogenesis, and multicellular branching tubulogenesis. In the present study, we demonstrate that HGF stimulates epithelial cells to express neutrophil gelatinase-associated lipocalin (Ngal), a member of the lipocalin family of secreted proteins that has recently been shown to participate in mesenchymal-epithelial transformation via its ability to augment cellular iron uptake. At concentrations below those found to mediate iron transport, purified Ngal can induce a promigratory and probranching effect that is dependent on ERK activation. The suppression of Ngal expression using short hairpin RNA results in increased cyst formation by tubular cells. However, the simultaneous addition of Ngal and HGF leads to direct association of the two proteins, and results in a partial inhibition of HGF-mediated activation of c-Met and the downstream MAPK and phosphatidylinositol 3-kinase signaling pathways. This inhibitory effect down-regulates HGF-stimulated single cell migration, and limits branching morphogenesis at both the single cell and multicellular level. These experiments demonstrate that the local expression of Ngal can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures while simultaneously negatively modulating the branching effects of HGF.

OBJECTIVE

The recruitment of lymphocytes to tissues via endothelium has been studied extensively but less is known about the signals that direct migration and positioning within tissues. Liver myofibroblasts associate with lymphocytes in hepatitis and are positioned below the sinusoidal endothelium, through which lymphocytes are recruited to the liver. We investigated whether activated human liver myofibroblasts (aLMF) affect the migration and accumulation of lymphocytes within the inflamed liver.

METHODS

The ability of human aLMF and hepatic stellate cells to promote lymphocyte chemotaxis, adhesion, and migration was studied in vitro.

RESULTS

When cultured in vitro, aLMF from diseased human liver and hepatic stellate cells from noninflamed liver secrete a distinct profile of cytokines comprising interleukin (IL)-6, IL-12, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and the chemokines CCL2, CCL3, CCL5, CXCL8, CXCL9, and CXCL10. aLMF-conditioned media had chemotactic activity for lymphocytes, which partially was inhibited by pertussis toxin. IL-6, HGF, and VEGF all contributed to G-protein-coupled receptor-independent chemotaxis of lymphocytes. Lymphocytes adhered to aLMF via intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and a proportion of adherent cells migrated through the fibroblast monolayer, mediated by IL-6, HGF, and VEGF.

CONCLUSIONS

Human aLMF support G-protein coupled receptor-dependent and -independent lymphocyte adhesion and migration and thereby regulate the recruitment and positioning of lymphocytes in chronic hepatitis.

Mesangial cell activation is a predominant pathologic feature of diabetic nephropathy that precedes the accumulation of extracellular matrix leading to glomerulosclerosis. For understanding the potential mechanism by which hepatocyte growth factor (HGF) ameliorates diabetic nephropathy, the effects of HGF on mesangial cell activation induced by TGF-beta1 were investigated. Western blot analysis and immunostaining revealed that HGF suppressed alpha-smooth muscle actin expression induced by TGF-beta1 in cultured rat and human mesangial cells. HGF also inhibited TGF-beta1-mediated fibronectin and type I collagen expression. Such action of HGF was dependent on the activation of extracellular signal-regulated kinase-1 and -2 but not on Akt and p38 mitogen-activated protein kinase. HGF did not affect TGF-beta1-mediated Smad2 phosphorylation and its nuclear translocation. However, it rapidly upregulated Smad transcriptional corepressor TG-interacting factor (TGIF) abundance in mesangial cells, which was primarily mediated by stabilizing its protein from degradation. Ectopic expression of TGIF markedly suppressed Smad-mediated activation of TGF-beta1-responsive promoter activity and completely blocked TGF-beta1-induced alpha-smooth muscle actin expression. In vivo, TGIF expression was dramatically downregulated in the glomeruli of diabetic kidneys, and delivery of exogenous HGF induced TGIF expression. These results suggest that HGF specifically antagonizes the profibrotic action of TGF-beta1 in mesangial cells by stabilizing Smad transcriptional corepressor TGIF.

We identified a naturally occurring hepatocyte growth factor (HGF) variant, whose predicted sequence extends only through the second kringle domain of this plasminogen-related molecule. This smaller molecule, derived from an alternative HGF transcript, lacked mitogenic activity but specifically inhibited HGF-induced mitogenesis. Cross-linking studies demonstrated that the truncated molecule competes with HGF for binding to the HGF receptor, which has been identified as the c-met protooncogene product. Thus, the same gene encodes both a growth factor and its direct antagonist.

The epidermal growth factor receptor (EGFR) is overexpressed and activated in many human cancers and predicts poor patient prognosis. Targeting the kinase domain with specific EGFR tyrosine kinase inhibitors (TKIs) like gefitinib and erlotinib has been used in anticancer treatments. However, patient response rates in different human cancers were initially low. Only a subgroup of non-small-cell lung cancer (NSCLC) patients harboring EGFR-activating mutations responds to EGFR TKI treatment, but most of these responders relapse and acquire resistance. Recent clinical studies have demonstrated that MET proto-oncogene overexpression correlates with resistance to EGFR TKI treatment. Similarly to MET overexpression, the tumor microenvironment-derived ligand hepatocyte growth factor (HGF) was shown to activate Met and thereby induce short-term resistance to EGFR TKI treatment in gefitinib-sensitive NSCLC cell lines in vitro. However, only little is known about the HGF/Met-induced EGFR TKI resistance mechanism in other human cancer types. Therefore, in order to develop possible new anticancer strategies for diverse human cancers, we screened 12 carcinoma cell lines originating from the breast, kidney, liver and tongue for HGF-induced EGFR tyrosine kinase (TK)-inhibition. In addition, in order to advance our understanding of a TK-inactive EGFR, we used EGFR co-immunoprecipitation, followed by mass spectrometry to identify novel HGF-induced EGFR binding partners, which are potentially involved in tyrosine kinase-independent EGFR signaling mechanisms. Here we show for the first time that HGF-induced EGFR TK-inhibition is a very common mechanism in human cancers, and that the kinase-inactive EGFR directly interacts with and stabilizes several cancer-relevant proteins, including the receptor tyrosine kinases Axl and EphA2, and the CUB domain-containing protein-1. This study has strong implications for the development of new anticancer strategies.

OBJECTIVE

Periodontal ligament (PDL) is a reliable cell source for periodontal regeneration. In this study, an optimal protocol for the extraction, expansion, and characterization of human PDL (hPDL) cells was examined for clinical trials.

METHODS

hPDL tissues were obtained from 41 surgically extracted teeth and digested with enzymes. Human adipose-derived stem cells (hADSCs), bone marrow-derived mesenchymal stem cells (hBMMSCs), and gingival fibroblasts (hGFs) were used for comparison. For each sample, the proliferative capacity, colony-forming ability, alkaline phosphatase activity, differentiation ability, the cell surface antigens, gene expression, and regenerative potential were examined.

RESULTS

hPDL cells were more successfully extracted with collagenase/dispase [29/30 (96.7%)] than with trypsin/EDTA [8/11 (72.7%)], and exhibited osteogenic potential both in vitro and in vivo. The proliferation of hPDL cells was rapid at a low cell density. hPDL cells frequently differentiated into cementoblastic/osteoblastic lineage (∼60%). In contrast, their adipogenic and chondrogenic potentials were lower than those of hADSCs and hBMMSCs. Some genes (NCAM1, S100A4, and periostin) were preferentially expressed in hPDL cells compared with those of hBMMSCs and hGFs. Immunohistochemical studies revealed the expressions of S100A4 and periostin in hPDL tissue.

CONCLUSIONS

A protocol for the successful cultivation and validation of hPDL cells is proposed for clinical settings.

Following liver regeneration after partial hepatectomy, liver grows back precisely to its original mass and does not exceed it. The mechanism regulating this "hepatostat" is not clear and no exceptions have been found to date. Although pathways initiating liver regeneration have been well studied, mechanisms involved in the termination of liver regeneration are unclear. Here, we report that integrin-linked kinase (ILK) (involved in transmission of the extracellular matrix [ECM] signaling by way of integrin receptors) and/or hepatic adaptations that ensue following ILK hepatocyte-targeted removal are critical for proper termination of liver regeneration. Following partial hepatectomy (PHx), mice with a liver-specific ILK ablation (ILK-KO-Liver) demonstrate a termination defect resulting in 58% larger liver than their original pre-PHx mass. This increase in post-PHx liver mass is due to sustained cell proliferation driven in part by increased signaling through hepatocyte growth factor (HGF), and the beta-catenin pathway and Hippo kinase pathways.

CONCLUSIONS

The data indicate that ECM-mediated signaling by way of ILK is essential in proper termination of liver regeneration. This is the first evidence of a defect leading to impaired termination of regeneration and excessive accumulation of liver weight following partial hepatectomy.

Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

Using PCR, hepatocyte growth factor (HGF) and c-met (HGF receptor) expression were analyzed in the developing mouse kidney. Both HGF and c-met were expressed from Gestational Day 11.5 onward, the time at which branching morphogenesis of ureteric bud occurs. Coculturing the embryonic kidney with MDCK cells seeded in Type I collagen induced branching morphogenesis of the MDCK cells. When a monospecific antiserum against HGF was included in the culture medium, the branching morphogenesis induced by the embryonic kidney was markedly inhibited (73%). This anti-HGF serum also inhibited metanephric growth when incubated with 11.5- to 12.5-day embryonic kidneys in an organ culture assay. No inhibition was observed by nonimmune serum. Perturbation of ureteric duct development as well as extraductal metanephric development was observed in embryonic kidneys incubated with the anti-HGF serum. Together, our data indicates an important role for HGF in kidney development.

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.

Receptor tyrosine kinases have come to fruition as therapeutic targets in a variety of malignancies. In this group of targets, the c-Met receptor tyrosine kinase plays an important role in increased cell growth, reduced apoptosis, altered cytoskeletal function, increased metastasis, and other biologic changes. The ligand for c-Met is hepatocyte growth factor (HGF), also known as scatter factor. Met is overexpressed and mutated in a variety of malignancies, among which germline mutations are of particular interest. Most mutations of Met have been found in the juxtamembrane, the tyrosine kinase, and the semaphorin domain. Met gain-of-function mutations lead to deregulated or prolonged tyrosine kinase activity, which is instrumental to its transforming activity. This review summarizes the biologic functions regulated by Met and its structural requirements as well as related developments in targeted therapy. Treatment approaches, including antagonism of HGF binding to Met, targeting of RNA and the Met protein, and inhibition of the tyrosine kinase domain of Met, are highlighted. Targeting of the HGF/Met pathway, alone or in combination with standard therapies, is likely to improve current therapies in Met-dependent malignancies.

OBJECTIVE

Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes.

METHODS

The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses.

RESULTS

Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation.

CONCLUSIONS

IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver.

OBJECTIVE

To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors. Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes.

RESULTS

In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-beta. The TGF-beta is activated, upregulating expression of CTGF. Under the influence of TGF-beta and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury.

CONCLUSIONS

HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.

Mesenchymal stromal cells (MSC) can be expanded from different sources. We compared the influence of inflammation and TLR ligation on the phenotype and function of MSC derived from bone marrow (BM), adipose tissue (AT), and Wharton's jelly (WJ). WJ-MSC were featured by a lack of TLR4 expression. While inflammation upregulated TLR3 in all three MSC types, TLR4 upregulation was observed only on BM-MSC. TLR ligation increased the production of inflammatory cytokines in BM- and AT-MSC but not in WJ-MSC and augmented anti-inflammatory cytokines in AT-MSC. Although inflammation increased in all MSC types the secretion of inflammatory cytokines, additional TLR triggering did not have further effect on WJ-MSC. The immunosuppressive potential of WJ-MSC on MLR was affected neither by inflammation nor by TLR triggering. This resistance was related to an overproduction of HGF. These data indicate that MSC source could be of importance while designing immunomodulating cell therapy in transplantation.

OBJECTIVE

Mesenchymal stem cells (MSCs) may modulate inflammatory responses resulting in improvement in inflammatory diseases, as well as tissue regeneration via cellular differentiation. We examined the therapeutic effects of exogenously administered MSCs in dextran sulfate sodium (DSS)-induced colitis in rats.

METHODS

Experimental colitis was produced in inbred male Lewis rats by administration of 4% DSS in drinking water for 7 days. MSCs (5x10(6) cells) which were isolated from whole marrow cells and cultured in an optimal medium for MSC outgrowth were administered to the treated rats via the tail vein on days 0, 2, and 4. On day 7, we evaluated colon length, histological changes, and colonic various mRNA expressions by RT-PCR. Localization of MSCs was evaluated using a green-fluorescent cell linker dye. To evaluate the anti-inflammatory action of MSCs, we assayed LPS-induced TNF-alpha secretion in a co-culture of MSCs and monocytes (THP-1 cells) using ELISA.

RESULTS

MSCs reduced in bloody stools, weight loss, colon shortening, and microscopic injuries. In the rectum of MSCs-treated rats, mRNA expression of TNF-alpha, IL-1beta, and COX-2 decreased to 40, 15, and 15% of their respective control levels. MSCs significantly suppressed mRNA expression of VEGF, HGF, and b-FGF to 40, 25, and 25% of their respective control levels. Green-fluorescent-labeled MSCs were found only within the lamina propria in inflamed regions. LPS-induced TNF-alpha secretion by THP-1 cells was significantly suppressed by co-culture with MSCs dose-dependently.

CONCLUSIONS

We conclude that exogenous MSCs accumulated in inflamed tissues and ameliorated DSS-induced colitis via a local anti-inflammatory action.

Sepsis and endotoxemia are involved in the development of fulminant hepatic failure, the prognosis of which is extremely poor and the mortality is high, with no available effective therapy. Here, we report that hepatocyte growth factor (HGF) exerts potent antiapoptotic effects in vivo and effectively prevents endotoxin-induced fulminant hepatic failure in mice. The animals were intraperitoneally injected three times with 120 micrograms human recombinant HGF or saline 6 hours and 30 minutes before and 3 hours after an intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (GalN). Administration of LPS + GalN, without HGF, rapidly led to massive hepatocyte apoptosis and severe liver injury, and all mice died of hepatic failure within 8 hours. In contrast, administration of human recombinant HGF strongly suppressed extensive progress of hepatocyte apoptosis and the liver injury induced by LPS + GalN, and 75% of the HGF-treated mice survived. Moreover, HGF strongly induced Bcl-xL expression and blocked apoptotic signal transduction upstream of CPP32 (caspase-3) in the liver, thereby leading to inhibition of massive hepatocyte apoptosis. We suggest that HGF may well have the potential to prevent fulminant hepatic failure, at least through its potent antiapoptotic action.

Overexpressed or activated hepatocyte growth factor receptor, encoded by the MET proto-oncogene, was found in the majority of colorectal carcinomas (CRCs), whose stepwise progression to malignancy requires transcriptional activation of beta-catenin. We here demonstrate that a functional crosstalk between Met and beta-catenin signaling sustains and increases CRC cell invasive properties. Hepatocyte growth factor (HGF) stimulation prompts beta-catenin tyrosine phosphorylation and dissociation from Met, and upregulates beta-catenin expression via the phosphatidylinositol 3-kinase pathway in conditions that mimic those found by the invading and metastasizing cells. Additionally, a transcriptionally active form of beta-catenin, known to be oncogenic, enhances Met expression. Furthermore, HGF treatment increases the activity of the beta-catenin-regulated T-cell factor transcription factor in cells expressing the wild-type or the oncogenic beta-catenin. In the mirror experiments, either Met or beta-catenin knocking down also reduces their protein level. In biological assays, beta-catenin knocking down abrogates the HGF-induced motile phenotype, whereas active beta-catenin fosters ligand-independent cell scattering. Met and beta-catenin also cooperate in promoting entry into the cell cycle and in protecting cells from apoptosis. In conclusion, Met and beta-catenin pathways are mutually activated in CRC cells. This might generate a self-amplifying positive feedback loop resulting in the upregulation of the invasive growth properties of CRC cells.

Interstitial myofibroblasts are alpha-smooth muscle actin-positive cells that play a crucial role in the accumulation of excess extracellular matrix during renal interstitial fibrogenesis. Despite their importance in the pathogenesis of renal fibrosis, relatively little is known about the regulators and the mechanism controlling the activation of renal interstitial myofibroblasts in disease conditions. Here, we show that hepatocyte growth factor (HGF) acts as a potent inhibitor of the transforming growth factor (TGF)-beta1-mediated myofibroblastic activation from normal rat renal interstitial fibroblasts (NRK-49F). Simultaneous incubation of HGF abolished TGF-beta1-induced de novo alpha-smooth muscle actin expression, F-actin reorganization, and interstitial collagen I overproduction in a dose-dependent manner. To decipher the mechanism underlying HGF antagonizing TGF-beta1's action, we examined the effects of HGF on TGF-beta1-mediated Smad signaling. HGF neither inhibited Smad-2/3 phosphorylation and their association with Smad-4 induced by TGF-beta1, nor significantly affected inhibitory Smad-6 and -7 expression and cellular abundance of Smad transcriptional co-repressors in NRK-49F cells. However, pretreatment with HGF markedly attenuated activated Smad-2/3 nuclear translocation and accumulation. This action of HGF was apparently dependent on HGF-mediated extracellular signal-regulated kinase-1 and -2 (Erk-1/2) phosphorylation and activation. Inhibition of Erk-1/2 activation by Mek kinase inhibitor PD98059 restored TGF-beta1-mediated Smad-2/3 nuclear accumulation and myofibroblast activation. In vivo, HGF selectively blocked Smad-2/3 nuclear accumulation in renal interstitial cells in the fibrotic kidneys induced by unilateral ureteral obstruction. Therefore, HGF suppresses TGF-beta1-mediated renal interstitial myofibroblastic activation; and this action of HGF is likely related to a mitogen-activated protein kinase-dependent blockade of Smad nuclear translocation.

The receptor tyrosine kinase, c-MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) have become leading candidates for targeted cancer therapies. Inappropriate c-MET signaling through autocrine, paracrine, amplification, and mutational activation occurs in virtually all types of solid tumors (http://www.vai.org/met), contributing to one or a combination of proliferative, invasive, survival, or angiogenic cancer phenotypes. c-MET and HGF/SF participate in all stages of malignant progression and represent promising drug targets in a variety of cancer types, including carcinomas, sarcomas, and brain tumors. While many are in pre-clinical testing, a few inhibitors have entered clinical trials. With hundreds of thousands of potential responding cancers that express c-MET, the interest in this molecule as a drug target is not surprising. However, the cognate c-MET diagnostic tests lag behind. In addition, despite the great enthusiasm based on response rates in phase I trials, there is a need for caution. It is almost without question that combination therapies with c-MET-HGF/SF inhibitors will be required for most cancers to achieve a cytotoxic tumor response.

Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous, non-embryonic origin. The purpose of this study was to establish efficient protocols for lineage specification of human iPS cells into functional glucose-responsive, insulin-producing progeny. We generated human iPS cells, which were then guided with recombinant growth factors that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness factors, human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions, fate specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm, followed by priming with fibroblast growth factor 10 (FGF10) and KAAD-cyclopamine. Addition of retinoic acid, boosted by the pancreatic endoderm inducer indolactam V (ILV), yielded pancreatic progenitors expressing pancreatic and duodenal homeobox 1 (PDX1), neurogenin 3 (NGN3) and neurogenic differentiation 1 (NEUROD1) markers. Further guidance, under insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), was enhanced by glucagon-like peptide-1 (GLP-1) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX1, and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-1 offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny, refining the development of a personalized platform for islet-like cell generation.